Scientia Agricultura Sinica

Overexpression of OsPR1A Enhanced Xa21-Mediated Resistance to Rice Bacterial Blight

LIU YuQing,YAN GaoWei,ZHANG Tong,LAN JinPing,GUO YaLu,LI LiYun,LIU GuoZhen,DOU ShiJuan

Scientia Agricultura Sinica. 2021, 54(23): 4933-4942. doi:10.3864/j.issn.0578-1752.2021.23.001

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【Background】Previous studies revealed that the expression of pathogenesis-related protein OsPR1A was regulated by the upstream resistance gene Xa21. The rapid induction of OsPR1A protein at early stage after inoculation was crucial in Xa21-mediated rice bacterial blight resistance. The expression of OsPR1A was induced by Xanthomonas oryzae pv. oryzae (Xoo). OsPR1A was well known as a marker gene to demonstrate the reaction between rice and pathogen, however, no direct evidence was obtained for the biological function of OsPR1A. 【Objective】 In this paper, transgenic plants overexpression OsPR1A were obtained and the phenotype and agronomic traits were investigated. The relationship between OsPR1A expression and resistance were surveyed to further explore the function of OsPR1A in the process of rice resistance to bacterial blight.【Method】The construct of OsPR1a-OX was transferred to rice recipient 4021 via Agrobacterium-mediated protocol. Positive homozygous transgenic lines were identified by PCR and western blot (WB) respectively. At the mature stage, the phenotype and agronomic traits of OsPR1A overexpression rice plants were investigated, including plant height, spike length, tiller number, seed setting rate and grain size. Rice seedlings of TP309, 4021 and OsPR1A overexpression plants grown for two weeks were inoculated with Xoo at 31℃. The length of lesions was measured at 0, 2, 4, 6, 8, 10, and 12 days post-inoculation (dpi) respectively. At 0, 4 and 6 dpi, the rice leaves of TP309, 4021 and OsPR1A overexpression plants were collected to extract total protein, and the expression profiling of OsPR1A were surveyed by WB.【Result】The OsPR1a-OX transformation vector was constructed and transformed into recipient 4021. Two homozygous OsPR1A overexpression lines (#704 and #709) were identified. At the mature stage, the phenotype and agronomic traits of the OsPR1A overexpression plants were investigated. Compared with the control 4021, #704 and #709 lines showed lower plant height, shorter panicle length, fewer tiller number, lower seed-setting rate. The grain size in transgenic rice plants were larger, which might be related to the lower seed-setting rate. At 31℃, the lesion length of OsPR1A overexpression plants was significantly shorter than that of the control 4021 (P<0.05). At 0, 4, and 6 dpi, the abundance of OsPR1A expression of overexpression plants was higher than that of 4021 and TP309, and the high level of OsPR1A protein might contribute to the resistance of Xoo.【Conclusion】OsPR1A overexpression transgenic plants were obtained by the Agrobacterium-mediated method. Overexpression of OsPR1A affected the normal development of rice plants and also enhanced the resistance to bacterial blight mediated by Xa21.

Effects of Physicochemical Properties of Wheat (Triticum aestivum L.) Starch with Different HMW-GSs Combinations on Dough Stability

NIU HongZhuang,LIU Yang,LI XiaoPing,HAN YuXuan,WANG KeKe,YANG Yan,YANG QianHui,MIN DongHong

Scientia Agricultura Sinica. 2021, 54(23): 4943-4953. doi:10.3864/j.issn.0578-1752.2021.23.002

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【Objective】Wheat dough is made of starch wrapped around filaments of gluten proteins. The rheological properties of dough have influence on the processing qualities of food. To explore the effect of starch physicochemical properties on the rheological properties of dough can provide a theoretical basis for clarifing the function of starch in dough.【Method】12 wheat varieties or strains were used as experimental materials, and the 12 test materials had three high molecular weight glutenin subunits (HMW-GSs) combinations. The subunits of Xinmai 26, Jimai 44, Xinong 615, and Gaocheng 8901 were 1, 7+8, and 5+10; the subunits of Xinong 221, Xinong 979, Xinong 633, and Xinong 059 were 1, 7+8, and 2+12; the subunits of 15(85)2A, 14(417)0-0-10, Xiaoyan 22, and Zhoumai 18 were 1, 7+9, and 2+12. Starch was extracted from the seeds of the test materials and the morphology of starch grains was observed by scanning electron microscope. The physicochemical properties of starch (including starch grain distribution, amylose content, relative crystallinity, short-range order degree, and thermal characteristics) and dough mixograph factors (including formation time and stability time) were determined and analyzed.【Result】For the starch granules of the 12 materials, the large starch granules were irregular oval, while the small starch granules were irregular oval or polyhedron. The starch grains of Xinmai 26, Jimai 44, Gaocheng 8901, Xinong 221, Xinong 979, and Xinong 059 were closely arranged, while the starch grains of Xinong 615, Xinong 633, 15(85)2A, 14(417)0-0-10, Xiaoyan 22, and Zhoumai 18 were dispersed. Xinong 979, 15(85)2A, Xiaoyan 22, and Zhoumai 18 had larger diameter of A-type starch grains. The higher the content of A-type starch grains, the lower the content of B-type starch grains, and the smaller the ratio of B-type starch grains to A-type starch grains. When the amylose content between two materials was similar, their dough stability time was similar. The X-ray diffraction patterns of the starches of the 12 materials are similar and all belong to A-type crystal structure. The Fourier transform infrared spectrum analysis of starch showed that Xinmai 26 had the highest 1 045/1 022 cm^-1 value and the lowest 1 022/995 cm^-1value, while the value of 1 022/995 cm^-1 in Zhoumai 18 was the highest, but the value of 1 045/1 022 cm^-1 was lower. This indicates that a higher 1 045/1 022 cm^-1 value of the test material usually corresponds to a lower 1 022/995 cm^-1 value. The starch properties of the 12 test materials were different, and the stability time of the dough was also significantly different. The materials Xinmai 26, Jimai 44, Xinong 221, Xinong 979, and Xinong 633 had longer dough stability time, and the dough stability time of Xinmai 26 was much longer than that of other materials.【Conclusion】 Among the 12 test materials, amylose content (r=0.88, P<0.01) and short-range order of starch (r=0.83, P<0.01) were significantly positively correlated with the dough stability time. The A-type starch content (r=0.61, P<0.05), starch relative crystallinity (r = 0.84, P<0.01), and enthalpy of starch gelatinization (r=0.71, P<0.01) were significantly negatively correlated with the dough stability time.

Comprehensive Evaluation of Low-Fertility Tolerance of Different Sorghum Cultivars in Middle-Late-Maturing Area

ZHANG Yan,WANG JinSong,DONG ErWei,WU AiLian,WANG Yuan,JIAO XiaoYan

Scientia Agricultura Sinica. 2021, 54(23): 4954-4968. doi:10.3864/j.issn.0578-1752.2021.23.003

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【Objective】Soil fertility is essential to crop growth and yield. The arable land per capita in China is low, and approximately 21.95% of the arable land has serious barriers for crop production. To ensure food security, it is necessary to exploit cultivated land reserves, along with the efforts to increase the productivity of arable land. Therefore, it is important to cultivate crops that can tolerate low-fertility in marginal lands, to avoid land competition with other main crops and meet increasing food demand. To better use marginal land for sorghum production, a field experiment was conducted in Yuci, Shanxi Province in 2019, to study the variation in low-fertility tolerance ability of different sorghum cultivars. This study aimed to identify the sorghum cultivars with strong tolerance to low-fertility and the index to evaluate low-fertility tolerance, and the results would provide evidence for sorghum selection in marginal land cultivation.【Method】In this study, a field experiment including 23 sorghum cultivars was conducted with contrasting soil fertility treatments, including high soil fertility (control) treatment and low soil fertility treatment (stress). A total of 15 parameters including yield and yield components, dry matter accumulation of shoot and nutrient uptake were investigated. The low-fertility index of each parameter was calculated. Principal component analysis (PCA) and cluster analysis were used to evaluate low-fertility tolerance and identify low-fertility tolerant cultivar. Low-fertility index and comprehensive evaluation value were used, combined with regression analysis and correlation analysis, to identify the optimal indicators for low-fertility tolerance.【Result】The results showed that the yield, grain number per spike, harvest index, dry matter accumulation at harvest and after heading, leaf area index (LAI), panicle length and width of sorghum were decreased by the low-fertility stress, compared with those in high-fertility plots. The decreasing rate was as follows: dry matter accumulation after heading>yield>grain number per spike>dry matter accumulation of shoot>LAI>panicle width>harvest index. The accumulation of N, P, K in both grains and shoot above ground was also decreased. The N accumulation was the most sensitive to low-fertility stress. The 15 parameters were categorized into five comprehensive indexes (the cumulative contribution rate was 89.28%) by principal component analysis, and the comprehensive evaluation value (D) of the low-fertility tolerance was calculated. According to the D value and the cluster analysis, the 23 sorghum cultivars were divided into four tolerance levels. Six, seven, seven and three cultivars were classified as stronger (0.633≤D≤0.755), strong (0.467≤D≤0.592), weak (0.310≤D≤0.421) and weaker (0.166≤D≤0.246) tolerance to low fertility, respectively. The cultivars Jiniang 2, Liaoza 19, Jinza 18, Jinza 22, Jinfeng 301 and Jinza 28 had the highest D value. Stepwise regression analysis was used to establish the optimal regression equation to evaluate the sorghum low-fertility tolerance. The yield, dry matter accumulation of shoot, N accumulation above ground, grain P accumulation, grain K accumulation and panicle width were selected, which had significant effects on the tolerance to low-fertility stress. The low-fertility index of yield, dry matter accumulation of shoot, N accumulation in shoot above ground, P accumulation in shoot above ground, grain N accumulation and grain P accumulation were significantly correlated with D value. The correlation coefficients were 0.845, 0.836, 0.766, 0.778, 0.761 and 0.757, respectively. 【Conclusion】There were large variation in low-fertility tolerance of the 23 sorghum cultivars. The cultivars of Jiniang 2, Liaoza 19, Jinza 18, Jinza 22, Jinfeng 301 and Jinza 28 were significantly tolerant to low-fertility land. The low-fertility index of grain yield, dry matter accumulation of shoot, N accumulation above ground and grain P accumulation were recommended in rapid screening of low-fertility tolerant sorghum cultivars.

Canopy Population Quality Characteristics of Mechanical Transplanting Hybrid Indica Rice with “Reducing Hills and Stabilizing Basic-Seedlings” in Low-Light Region of Southwest China

TAO YouFeng,PU ShiLin,ZHOU Wei,DENG Fei,ZHONG XiaoYuan,QIN Qin,REN WanJun

Scientia Agricultura Sinica. 2021, 54(23): 4969-4983. doi:10.3864/j.issn.0578-1752.2021.23.004

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【Objective】The effects of reducing hills and stabilizing basic-seedlings (RHSB) on population canopy quality characteristics of mechanical transplanting indica hybrid rice were investigated in this study, which could provide theoretical support for the promotion and application of mechanical transplanting technology of hybrid rice in low-light paddy region of Southwest China. 【Method】 A two-factor random block design experiment with different rice varieties (Fyou 498 is a loose plant type, and Yixiangyou 2115 is a compact upper and drooping lower plant type at middle and late stages) and different basic seedlings rate (42×10⁴/hm² and 63×10⁴/hm²) was conducted to study the effects of different field collocation patterns (conventional field collocation (CFC) and RHSB) on the canopy architecture, photosynthetic characteristics, and microclimate environment (e.g. canopy temperature, humidity, and light transmittances) of mechanical transplanting hybrid rice population in 2016 and 2017. 【Result】(1) Compared with CFC, RHSB with a greater specific leaf weight of the upper three leaves (F_{second leaf}=23.67^** and F_{third leaf}= 16.91^** in 2017) could maintain a similar green leaf area per stem and grain/leaf ratio at heading stage. Compared with CFC, RHSB also led to the 23.84%, 23.53%, and 13.79% significant increase in photosynthetic rate, stomatal conductance, and transpiration rate of flag leaf at heading stage, respectively. (2) RHSB markedly decreased the canopy convergent index, but increased the canopy amplitude, which increased in light transmittance and ventilation of canopy. RHSB increased the angle of primary tillerings of Fyou 498 at booting stage and heading stage in 2016, but decreased the angle of primary tillerings of Yixiangyou 2115 in 2016 and 2017. (3) Correlation analysis indicated that daily average temperature and diurnal variations of temperature and humidity at booting stage were significantly or extremely significantly positively correlated with the specific leaf weight of the flag leaf and the 2nd leaf at heading stage, but significantly negatively correlated with plant convergent index at heading stage. Furthermore, the daily average temperature and diurnal variations of humidity at booting stage was significantly positively correlated with canopy amplitude at heading stage. Daily average temperature and diurnal variations of temperature at heading stage were significantly or extremely significantly negatively correlated with the angle of primary tillering at tilling stage, jointing stage, and 20 d after heading stage. However, the inverse relation was observed by daily average relative humidity. RHSB could optimize the canopy architecture of rice, which contributed to the increase in canopy temperature and diurnal the variations of temperature at booting and heading stages, and increase diurnal the variations of humidity at booting, heading and 20 d after heading stage, but decrease in the relative humidity.【Conclusion】As one of the main technology that improved the mechanization level of rice production in the low-light region of Southwest China, RHSB optimized the population canopy structure and light distribution of mechanical transplanting hybrid rice, which resulted in the increase in the temperature and humidity difference between day and night within the population, and reduced the relative humidity. This contributed to the improvement in the population quality and photosynthetic rate, and contributed to the increase in rice grain yield.

Effects of Elevated Atmospheric CO₂Concentration and Temperature on Photosynthetic Characteristics, Carbon and Nitrogen Metabolism in Flag Leaves and Yield of Winter Wheat in North China

ZONG YuZheng,ZHANG HanQing,LI Ping,ZHANG DongSheng,LIN Wen,XUE JianFu,GAO ZhiQiang,HAO XingYu

Scientia Agricultura Sinica. 2021, 54(23): 4984-4995. doi:10.3864/j.issn.0578-1752.2021.23.005

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【Objective】This study was conducted to clarify the response and acclimation process of winter wheat under elevated carbon dioxide (CO₂) concentration and rising temperature in North China, so as to provide the theory basis for wheat production under future climate change condition.【Method】Winter wheat (Triticum aestivum ‘Zhongke2011’) plants were subjected to elevated CO₂ concentration (ambient concentration +200 μmol·mol^-1) and rising temperature (ambient temperature +2℃) at open top climate chambers. The photosynthetic traits, carbon and nitrogen metabolism, biomass accumulation and yield formation of winter wheat in response to elevated CO₂ concentration and rising temperature were investigated. 【Result】The rising temperature shorten the whole growth period and the time from florescence to harvest of this wheat cultivar. The net photosynthesis rate (P_n) was enhanced at booting stage by 24.7%, but was not obviously changed in elongation and filling stages. However, the leaf fiber content, soluble protein content and nitrate reductase activity in filling stage, and kernels per spike, thousand seed weight, biomass, and yield in harvest stage decreased by 23.0%. Elevated CO₂ concentration increased P_n in the elongation and booting stage by 32.8% and 40.7%, respectively. Elevated CO₂ concentration also increased leaf carbohydrate content in the filling stage and spike number per unit area, and improved yield by 26.1%, although which induced photosynthesis acclimation at the late growth stage. Moreover, elevated CO₂ concentration increased the time from florescence to harvest of wheat plants for 2 days, and improved P_nby 25.54%, leaf soluble sugar content, fiber content, and starch content under rising temperature conditions. 【Conclusion】Elevated CO₂ concentration could offset the negative impacts of rising temperature on biomass accumulation and yield of wheat plants by increasing the time from florescence to harvest, net photosynthesis rate and carbon metabolism.

Analysis of Photosynthetic Characteristics of Hybrid Wheat at Seedling Stage and Its Use for Early Prediction of Strong Heterosis Combinations

LI JiangLing,YANG Lan,RUAN RenWu,LI ZhongAn

Scientia Agricultura Sinica. 2021, 54(23): 4996-5007. doi:10.3864/j.issn.0578-1752.2021.23.006

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【Objective】The aim of this study was to provide a theoretical basis for early heterosis prediction of hybrid wheat by analyzing photosynthetic characteristics of leaves of different hybrid wheat combinations and their parents at the seedling stage, so that it was easy to select hybrids with strong heterosis. 【Method】 Six hybrid wheat combinations and their parents, including two restorer lines and four male sterile lines, were used as materials to measure the photosynthetic characteristics of wheat leaves at the seedling stage in the field, including net photosynthetic rate (P_n), stomatal conductance (G_s) and intercellular CO₂ concentration (C_i), transpiration rate (T_r), water use efficiency (WUE), chlorophyll content (Chl.), Rubisco content and the relative expression of rbcL and rbcS genes encoding Rubisco subunits as well as their heterosis and correlation. The yield traits were investigated at the wheat maturity stage, including plant height, length of main spike, biomass per plant, spike number per plant, grains per spike, grain yield per plant, spikelets of main spike, thousand grain weight, and harvest index. And then, correlation between grain yield and their photosynthetic characteristics was analyzed. 【Result】 P_n, G_s and T_r of six hybrid wheat showed significant heterosis over high parents. The average heterosis of P_n over high parents was 15.4%, G_s was 21.3%, and T_r was 11.46%. The higher P_n and stronger heterosis of hybrids resulted from the restorer lines with higher P_n, while C_i and WUE of some hybrids showed negative heterosis. The number of spikes per plant, yield per plant and biomass per plant had greater heterosis over high parents, in which the number of spikes per plant performed the highest heterosis with 34.21% on average. Correlation analysis showed that P_n was significantly or super-significantly positively correlated with G_s, T_r, grain yield per plant and biomass per plant, suggesting that G_s and T_r could help to screen high photosynthetic wheat, and P_n at the seedling stage had important relationship with yield. Among them, the Rubisco activity of hybrid wheat was higher than that of its parents, and it had a significant heterosis over high parents, with an average heterosis of 5.38% over high parents. Compared with rbcL, rbcS had a higher over high-parent heterosis and over mid-parent heterosis. The chlorophyll content mainly showed a negative advantage, few over mid-parent heterosis, and very few showed over high-parent heterosis. There was no significant correlation between P_n and other photosynthetic characteristics.【Conclusion】 The results indicated that the leaf photosynthesis of hybrid wheat at the seedling stage showed significant over high-parent heterosis, especially P_n, G_s and T_r, and P_n was significantly positively correlated with the yield per plant and the biomass per plant, so the level of net photosynthetic rate could be one of the important traits for early prediction of yield potential of hybrid combination.

The Infectivity of Aspergillus flavus in Peanut

JING Dan, YUE XiaoFeng, BAI YiZhen, GUO Can, DING XiaoXia, LI PeiWu, ZHANG Qi

Scientia Agricultura Sinica. 2021, 54(23): 5008-5020. doi:10.3864/j.issn.0578-1752.2021.23.007

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【Background】Aspergillus flavus can easily infect peanut and other agricultural products, and the aflatoxin produced by A. flavus is highly toxic, teratogenic and carcinogenic, threatening human and animal health and causing significant agricultural economic losses.【Objective】Based on the previous research about the distribution of A. flavus in China, this study aims to further investigate the infection characteristics of A. flavus strains with different toxigenicity and geographical sources, so as to provide scientific basis for screening and breeding of peanut cultivars with resistance to A. flavus, as well as the early risk warning and control of aflatoxin contamination.【Method】A total of 102 strains of A. flavus isolated from different regions (early maturing peanut region in northeast China, large peanut region in north China, intercropping peanut region in spring and summer in Yangtze River, and double cropping peanut region in spring and autumn in south China) of China were identified as non-toxigenicity (ND), medium-low toxigenicity (0-1 500 μg·kg^-1) and high toxigenicity (>1 500 μg·kg^-1). The soaking method of the spores suspension was used to inoculate peanut seeds to identify the infection grade and infection index of A. flavus, and the infection difference of the strains and their correlation between different toxigenicity and different geographical sources were analyzed.【Result】The infection index of the 102 A. flavus strains to peanut was ranged from 3.89% to 67.50%. The proportion of A. flavus strains with intermediate or strong infectivity (the infection grade was 3 or 4, infection index>31%) accounted for 54.90%, and 18.63% of the total strains, mainly from Zhangshu in Jiangxi Province and Zhanjiang in Guangdong Province, were identified as intermediate or strong infectivity and high toxigenicity strains. Clustering and correlation analysis indicated that there was no significant correlation between the toxigenicity and infection index of A. flavus, however, the infectivity of toxigenic strains was significantly higher than that of the non-toxigenic strains. The infection index of medium-low toxigenicity and high toxigenicity strains accounted for the highest proportion in grade 4 and 3, respectively. Regional differences in the infectivity among the strains were analyzed. The results showed that average infection index of the strains from the south and the Yangtze River regions was 46.59% and 36.12%, respectively, and accounted for the highest proportion in grade 3 and 4. By contrast, the infection index of the strains from the northeast and northern regions was only 15.72% and 27.52%, respectively, and mainly distributed in grade 1 and 2. Strains from Guangdong Province, in the south China, had the highest average infection index (51.89%), while the average infection index of A. flavus in Liaoning Province, the northeast producing area, was the lowest, which was only 15.72%.【Conclusion】The infection characteristics of A. flavus and its relationship with toxigenicity and geographical sources were preliminarily clarified in this research. There was pathogenic differentiation among the strains, and the infectivity of the strains with different toxigenicity grades and geographical origins was significantly different. Strains with strong infectivity are mainly distributed in the south and Yangtze River regions of China. The results of this study are of great importance for the screening and breeding of peanut cultivars with resistance to A. flavus and the accurate early risk warning, prevention and control of aflatoxin contamination.

Sequence Analysis of Harmonia axyridis Pyruvate Kinase Gene and Its Regulation of Trehalose Metabolism

GE XinZhu,SHI YuXing,WANG ShaSha,LIU ZhiHui,CAI WenJie,ZHOU Min,WANG ShiGui,TANG Bin

Scientia Agricultura Sinica. 2021, 54(23): 5021-5031. doi:10.3864/j.issn.0578-1752.2021.23.008

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【Objective】Pyruvate kinase (PYK) is a regulatory glycolytic enzyme that regulates the balance between glycolysis and gluconeogenesis, so it has an important physiological role in insects. This study aims to explore the biological functions of HaPYK in Harmonia axyridis by analyzing the sequence structure of the three HaPYKs and RNA interference technology.【Method】Based on the three HaPYK sequences (named HaPYK1-1, HaPYK1-2, HaPYK2, respectively) obtained from the whole genome of H. axyridis, the physicochemical properties, sequence structure and homology with other insects through bioinformatics knowledge were analyzed. Then, the dsRNA synthesized in vitro was injected into the adult on the 1st day of emergence by microinjection. The experimental materials were collected 48 and 72 h after injection, and the total RNA extraction and reverse transcription experiments were performed. In order to evaluate the inhibitory effect, real-time fluorescent quantitative PCR (qRT-PCR) technology was used to determine the expression levels of three HaPYKs. Finally, the expression levels of genes related to trehalose metabolism, the content of glucose, glycogen, trehalose, and the activities of two trehalase enzymes were detected.【Result】The open reading frames of HaPYK1-1, HaPYK1-2 and HaPYK2 were 1 350, 1 569 and 1 656 bp, respectively. The protein sizes were 449, 522 and 551 aa, and the theoretical isoelectric points were 5.04, 5.02 and 5.01, respectively. The conserved domains predicted that the three HaPYKs belonged to the Pyruvate_kinase superfamily. The secondary structure of HaPYK was mainly α-helix and random coil, but also contained extended chain and β-corner. The oligomer types of the three sequences were all homologous tetramer. The phylogenetic tree showed that HaPYK was closely related to the same Coleoptera insects, such as Leptinotarsa decemlineata and Tribolium castaneum. After injection of dsHaPYK1-1, the expression levels of HaPYK1-2 and HaTRE1-4 were significantly down-regulated, while the expression levels of HaPYK2, HaTRE1-1, HaTRE1-2, HaTRE2-like, and HaTPS were significantly up-regulated; after injection of dsHaPYK1-2, the expression levels of HaTRE1-1, HaTRE1-2, HaTRE1-3, HaTRE1-5, HaTRE2, and HaTPS increased significantly; after injection of dsHaPYK2, only HaTRE1-1 was significantly up-regulated at 48 h, and the expression levels of HaTRE1-3, HaTRE1-4, HaTRE1-5, HaTRE2, HaTRE2-like were down-regulated. Compared with the control group, in the dsHaPYK1-2 group, the activity of TRE1 reduced significantly at 48 h, and the content of trehalose and glycogen increased significantly; in dsHaPYK1-1 or dsHaPYK2, the glucose content decreased at 48 h, the glucose and trehalose content increased at 72 h, and the enzyme activity of TRE2 increased significantly; at 48 h in dsHaPYK2 group, the trehalose content significantly reduced, while glycogen content significantly increased.【Conclusion】There are three HaPYKs in H. axyridis. By injecting exogenous dsHaPYK, the expression level of target genes can be successfully reduced. Suppressing their expression levels can affect the metabolism of trehalose, and the regulatory mechanisms of the three HaPYKs are not the same.

Effects and Mechanism of Humic Acid in Humic Acid Enhanced Phosphate Fertilizer on Fertilizer-Phosphorus Migration

JING JianYuan,YUAN Liang,ZHANG ShuiQin,LI YanTing,ZHAO BingQiang

Scientia Agricultura Sinica. 2021, 54(23): 5032-5042. doi:10.3864/j.issn.0578-1752.2021.23.009

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【Objective】The difference between HA (raw humic acid) and PHA (humic acid which extracted from humic acid enhanced phosphate fertilizers, HAP) on fertilizer-phosphorus migration, Ca²⁺ and phosphate adsorption characteristics was systematically compared to provide the theoretical basis for the study on high-efficiency mechanism of humic acid enhanced phosphate fertilizer. 【Method】 In this study, HA, HAP, and conventional phosphate fertilizer were prepared in the laboratory, and PHA was extracted by adjusting the pH of HAP’s solution based on the method of alkali-extraction acid-precipitation. The addition of HA or PHA accounted for 0.5% and 5% of the application amount of phosphate fertilizer, and marked with 0.5HA+P, 0.5PHA+P, 5HA+P, and 5PHA+P, respectively. Only phosphate fertilizer application (P) and no fertilizer application (CK) were arranged at the same time. Then, the effects of HA and PHA applicated with phosphate fertilizer on the migration of fertilizer-phosphorus in soil was investigated. In addition, the adsorption characteristics of HA or PHA on Ca²⁺ and phosphate were studied to reveal the mechanism that HA and PHA showed different performance on phosphorus migration. 【Result】Both HA and PHA could promote the migration of fertilizer-phosphorus. Phosphorus could migrate to 42 mm vertical distance from the fertilizer layer under the treatment of P. However, when phosphate fertilizers application combined with HA and PHA, it could reach 46 mm and 50 mm away from the fertilizer layer, respectively. This result was due to the fact that HA or PHA had a higher adsorption capacity on soil Ca²⁺, while the application of HA or PHA reduced phosphorus fixation. The cumulative percentage of soil available P in total P application tended to be stable after 42 mm away from the fertilizer layer, and 0.5 HA+P ≈ 5PHA+P>5HA+P>0.5PHA + P>P. The promotion effect of PHA on fertilizer-phosphorus migration would be enhanced with the addition of PHA increase, while HA was the opposite, which might be related to the stronger mobility and the weaker phosphate adsorption of PHA than that of HA. The adsorption of Ca²⁺ by HA and PHA was the result of membrane diffusion and intra-particle diffusion. However, the difficulty of Ca²⁺ diffusion to the surface of PHA particles was lower than that of HA, and the difficulty of Ca²⁺ diffusion in the interior of PHA particles was higher than that of HA. Langmuir isothermal adsorption model could well fit the isothermal adsorption curves of HA or PHA on Ca²⁺, and the theoretical maximum adsorption capacity of HA on Ca²⁺ was higher than that of PHA, but the adsorption of PHA on Ca²⁺ was mainly chemical adsorption.【Conclusion】Both HA and PHA had certain ability to adsorb Ca²⁺, so they could promote the migration of fertilizer-phosphorus in the soil. However, the migration distance of fertilizer-phosphorus of PHA was longer than that of HA, when phosphate application was combined with HA or PHA. This might be one reason why PHA could improve the use efficiency of phosphate fertilizer.

Effects of Straw Returning and Nitrogen Application Rate on Grain Yield and Nitrogen Utilization of Winter Wheat

WANG XinYuan,ZHAO SiDa,ZHENG XianFeng,WANG ZhaoHui,HE Gang

Scientia Agricultura Sinica. 2021, 54(23): 5043-5053. doi:10.3864/j.issn.0578-1752.2021.23.010

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【Objective】A seven-year location-fixed field experiment was carried out to explore the effects of combining straw returning with chemical nitrogen (N) fertilizer on wheat yield, grain protein content, aboveground N uptake, soil nitrate-N (NO₃^--N) residue at harvest, and apparent N balance in winter wheat-summer maize rotation region of Shaanxi, so as to provide a reference for yield increase and N utilization.【Method】The experiment was arranged in a split block design with two main treatments and five subplots. The main treatments included maize straw returning to soil (straw returning) and removal straw from field (control), and the subplots included five N application rate, i.e., 0 (N0), 84 kg·hm^-2 (N84), 168 kg·hm^-2 (N168), 252 kg·hm^-2 (N252), and 336 kg·hm^-2 (N336).【Result】For wheat yield, there was no significant difference between straw returning and control. Compared with N0, applying fertilizer N (including N84, N168, N252, and N336) increased grain yield by 18%-29%. However, compared with N168, there was a risk of yield reduction under high N application rate (N336). Straw returning and N application rate had an interactive effect on wheat yield. Compared with the control, the straw retuning increased grain yield by 5%-6% when N application rates were 252 and 336 kg·hm^-2, which was mainly due to the 5%-7% increase in the number of spikes. For grain protein, there was no significant difference between straw returning and control. Compared with N0, applying fertilizer N increased grain protein concentration by 16%-33%. For aboveground N uptake, there was no significant difference between straw returning and control. Compared with N0, applying fertilizer N increased aboveground N uptake by 36%-72%. Straw returning and N application rate had an interactive effect on aboveground N uptake. Compared with the control, the straw retuning increased aboveground N uptake by 5%-8% when N application rates were 252 and 336 kg·hm^-2. Compared with control, the straw returning increased the residual soil nitrate nitrogen by an average of 18%, and the increased nitrate nitrogen content was mainly distributed in the 70-170 cm soil layer. For N168 treatment, soil N was in the state of depletion in control, and soil N depletion was effectively compensated when straws were returned to the field. A further increase in N application rate would greatly increase N surplus, resulting in a larger environmental risks. Compared with control, applying fertilizer N had a greater contribution to N surplus.【Conclusion】Straw returning and applying fertilizer N had the ability to increase wheat yield and aboveground N uptake, while also increased residual NO₃^--N in soil and N surplus. Taking into account wheat yield, soil NO₃^--N residue, and apparent N balance, the strategy of straw returning combined with 168 kg·hm^-2 fertilizer N was beneficial to maintain wheat yield and to protect ecological environment.

Effects of Long-Term Reduce/Zero Tillage and Nitrogen Fertilizer Reducing on Maize Yield and Soil Carbon Emission Under Fully Plastic Mulched Ridge-Furrow Planting System

ZHOU YongJie,XIE JunHong,LI LingLing,WANG LinLin,LUO ZhuZhu,WANG JinBin

Scientia Agricultura Sinica. 2021, 54(23): 5054-5067. doi:10.3864/j.issn.0578-1752.2021.23.011

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【Objective】 The effects of tillage practices on soil respiration, carbon emission and crop yield under nitrogen reduction were clarified, and the relationship between maize growth and soil carbon emission was revealed.【Method】The long-term tillage practices and reduced fertilization experiment initiated in 2012 within two-year (2018-2019) was conducted at the Rainfed Agricultural Experimental Station of the Gansu Agricultural University in the Gansu province of northwestern China. This experiment is based on the technology of full-film double-ridge and furrow sowing maize with good effect of collecting rainfall and inhibiting evaporation and increasing temperature and soil moisture. The experiment adopted the split plot design, and the main plots were four tillage practices (conventional tillage, rotary tillage, subsoiling, and no-tillage) and the subplot were two nitrogen application levels (nitrogen reduction (200 kg·hm^-2) and conventional nitrogen application (300 kg·hm^-2)). Based on this experiment, the maize growth, soil respiration rate, carbon emission and soil organic carbon content, carbon emission efficiency (CEE) and net ecosystem productivity (NEP) were assessed.【Result】(1) Tillage practice and nitrogen application level significantly affected the growth of maize, and the effect of tillage practice on dry matter accumulation was mainly in the filling stage and maturity stage. No tillage treatment significantly improved the dry matter accumulation, growth rate and net assimilation rate at these stages, which increased grain yield by 2%-15% compared with other tillage practices; nitrogen application level had a greater effect on dry matter during jointing flowering stage, but the same effect was observed, and there was no significant difference in yield between N1 and N2 under the same tillage practice. (2) The soil respiration rate showed a single-peak curve that first increased and then decreased, reaching its peak in the big bell mouth-flowering period. The effects of tillage practices on soil respiration, carbon emissions and carbon emission efficiency were greater than the nitrogen levels. Compared with rotary tillage, tillage and subsoiling, no tillage decreased soil respiration rate by 4.3%, 12.9% and 24.3%, respectively, and total carbon emission decreased by 21.5%, 13.4% and 31.2%, respectively, while carbon emission efficiency increased by 26.5%-55.9%. Compared with other treatments, no tillage combined with nitrogen reduction reduced total nitrogen and carbon emission by 489-1917.5 kg·hm^-2, while the carbon emission efficiency increased by 20.1%-56.2%. (3) All treatments showed a “sink” of atmospheric CO₂, but no-tillage and reduced nitrogen fertilizer showed a stronger carbon sink effect. The organic carbon content in 0-5 cm soil layer was significantly increased by 11.3% (P<0.05) compared with conventional tillage; the organic carbon content in 0-10 cm soil layer was increased by 5.8% (P<0.05) compared with conventional tillage. (4) There was a significant positive correlation between the efficiency of carbon emissions and the accumulation of dry matter, the rate of growth and net assimilation rate, and a significant negative correlation between the efficiency of carbon emissions and the organic carbon of the soil. This was mainly because the cultivation practices and application of nitrogen promoted maize’s photosynthetic ability, obtained more CO₂, and enhanced maize’s capacity for carbon fixation. 【Conclusion】 Under the condition of 472-491 mm annual precipitation, no tillage combined with nitrogen reduction (200 kg·hm^-2) could improve maize yield, improve soil organic carbon content, reduce total carbon emission, and improve carbon emission efficiency. A green yield-increasing technology of full-film double-ridge and furrow sowing maize in the Loess Plateau of Longzhong was recommended to be used in production.

Identification, Cloning, and Expression Characteristics Analysis of Fe-S Cluster Assembly Genes in Grape

ZHANG Lu,ZONG YaQi,XU WeiHua,HAN Lei,SUN ZhenYu,CHEN ZhaoHui,CHEN SongLi,ZHANG Kai,CHENG JieShan,TANG MeiLing,ZHANG HongXia,SONG ZhiZhong

Scientia Agricultura Sinica. 2021, 54(23): 5068-5082. doi:10.3864/j.issn.0578-1752.2021.23.012

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【Objective】The aim of this study was to isolate and characterize grape Fe-S cluster assembly genes. The tissue-specific expression characteristics and differential response to iron depletion at the transcriptional level were analyzed to screen the dominant and candidate Fe-S cluster assembly genes in grape. 【Method】The Fe-S cluster assembly genes were screened and identified in grape genome by homologous cloning. The detailed characteristics of Fe-S cluster assembly genes and their encoded proteins in grape were analyzed by using a variety of bioinformatics software. The expression patterns of Fe-S cluster assembly genes in different tissues of grape and their corresponding responses to iron deficiency stress were analyzed by using real-time fluorescent quantitative PCR. The phylogenetic trees of ISU1 homologous proteins from different plants were established by using MEGE 7.0 software. 【Result】 In total, 46 Fe-S cluster assembly genes were retrieved and cloned from grape, which were located on a total of 16 chromosomes, containing 1-21 introns with different lengths. Grape Fe-S cluster assembly genes were mainly distributed in plastid, mitochondrion and cytosol, containing 14, 21 and 11 genes, respectively. Grape Fe-S cluster assembly proteins were found with a variety of subcellular structures. Moreover, the subcellular localization of Fe-S cluster assembly proteins with different assembly mechanisms was quite distinct. The sequence identity of ISU1 homologous proteins from 10 plant species was as high as 77%. Phylogenetic tree analysis indicated that ISU1 members belonging to the same genus, such as Arabidopsis and the llungiella of Cruciferae, rice and Brachypodium of Gramineae, and peach and apple of Rosaceae, were tended to be closely clustered together, while grape ISU1 was closely clustered with tomato ISU1. The expression levels of Fe-S cluster assembly genes were different among different tissues of 3-year-old adult tree and tissue culture seedling of Marselan grape, and the differences were significant. In particular, the expression level of ISU1 gene was the most abundant (especially in the mature fruit), followed by HSAC1, ISA2, NFU2, SUFA and SUFB, while the expression levels of SUFE2, NFS1, HSCA2, HSCA6, TAH18 and CIA2 were not detected in tested tissues of grape. Grape Fe-S cluster assembly genes were more sensitive to iron deficiency treatment, and all genes responded to iron deficiency treatment in at least one detected tissue sample. Notably, the expression level of 22 Fe-S cluster assembly genes were significantly regulated by iron deficiency in all tested tissues, and the expression levels in roots were likely to be up-regulated by iron deficiency, while the expression levels in shoots (stems and leaves) were prone to be down-regulated. 【Conclusion】46 Fe-S cluster assembly genes were cloned and identified in grape, which were located in plastid, mitochondrion and cytosol, respectively. The expression levels of Fe-S cluster assembly genes were significantly distinct among different tissues of 3-year-old adult tree and tissue culture seedlings, while the transcription levels in different seedlings tissues were significantly different in response to iron deficiency. The overall expression level of ISU1 gene was the highest in all tested tissues of grape. The genetic evolution distance between grape and tomato ISU1 homologous proteins was the closest.

Identification of Bud Sport Mutation of Satsuma Mandarin by Target SSR-seq Technology

HU DongMei,JIANG Dong,LI YongPing,PENG Lei,LI DongYun,ZHU YanSong,YANG YunGuang

Scientia Agricultura Sinica. 2021, 54(23): 5083-5096. doi:10.3864/j.issn.0578-1752.2021.23.013

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【Objective】 Most citrus cultivars especially some seedless varieties were originated from bud sports mutation. The plant morphological traits of these bud sports were susceptible to environment and cultivation conditions, so discriminating the mutant lines from its parents have been still hard tasks although many molecular marker approaches were developed. As the use of deep sequencing technology could simultaneously genotype multiple loci of the mutation lines, it was already widely used to discriminate citrus bud sports in high efficiency. In addition, the technical approach could also benefit to accurate the identification of citrus genetic accessions, the genetic diversity research and the variety rights protection. 【Method】 In this study, the Target-SSR sequencing approach was adopted to discriminate citrus bud sport. The SSR loci were firstly discovered by scanning of the clementine mandarin (Citrus reticulata Blanco) reference genome and satsuma mandarin (Citrus unshiu Macf.) genome sequences, and those high polymorphic SSR loci were used to design primers to distinguish 22 citrus accessions which including some bud sports materials. Finally, multiplexed PCR production obtained through 18 runs PCR amplification with 77 pair primers on two bud sports materials were used to construct target SSR sequencing library, and sequenced with MiSeq apparatus to genotype SSR and SNP variation. In addition, the genetic diversity of 22 citrus accessions were also analyzed by using the developed SSR markers in this study. 【Result】 69 101 SSRs and 80 193 SSR were obtained respectively from clementine reference genome and satsuma genome by using GMATA software, among which AT/TA motif SSR were the most, and 3 motifs AAT rank the second. The high polymorphic SSR loci and its flanking sequences were extracted to design primers for bud sports discrimination and library construction. Four pairs of SSR primers could accurately distinguish 22 citrus germplasms. Target SSR-sequencing with MiSeq apparatus not only simultaneously genotype multiple SSR loci at one time but also accurately identified the variant loci of SSRs. Combining with the identified SSR and SNP genotypes, it could be effectively distinguished two satsuma bud sports. 【Conclusion】In this research, an efficient SSR loci discovering method was developed, combining with target SSR multiplex amplification and deep sequencing on MiSeq platform, which could effectively discriminate citrus bud sports. This study not only provided an important means to identify citrus buds sports, but also facilitated the protection of citrus varieties and the management of citrus germplasms genetic diversity in the future.

Correlation Analysis of Auxin Involved in the Process of Petal Abscission of Tree Peony Luoyanghong Cut Flowers by Ethylene Promoting

YE Di,SHI Jiang,GAO ShuangCheng,WANG ZhanYing,SHI GuoAn

Scientia Agricultura Sinica. 2021, 54(23): 5097-5109. doi:10.3864/j.issn.0578-1752.2021.23.014

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【Objective】The aim of this study was to explore the physiological mechanisms of petal wilting and abscission during the senescence of cut tree peony flowers, so as to provide the theoretical basis for better development of storage and preservation technology of cut tree peony flowers.【Method】The cut flowers were pretreated with deionized water (CK), 20 µL·L^-1 ethylene antagonist Correxian (AVB), 20 µL·L^-1 ethylene releasing agent ethephon (CEPA), and 20 µL·L^-1 Correxian for 1 h, respectively, followed by 20 µL·L^-1 ethephon 1 h (AVB+CEPA). The flowers were then placed in individual vases filled with distilled water. The morphology of the petals absciss zone cells were observed by using a microscope. The petal anti-falling ability was determined by using the tensile test during the vase cutting process. The physiological effects of different pretreatments were observed respectively using the physiological and biochemical methods to determine the ethylene and auxin metabolism, the absciss zone hydrolysis-related enzyme activities, and the relative expressions of PsETR1, PsCTR1, PsEIN3, PsERF1, PsYUCCA10, PsPIN1, PsPME1, PsPG1 and PsBG1 genes. 【Result】 The results showed that the basal petal of the cut tree peony Luoyanghong had a clear separation layer structure. AVB significantly delayed the occurrence of ethylene climacteric in cut flowers, and decreased the ethylene release peak by 34.9% (P<0.05), while the petal anti-falling ability was improved. The activities of polygalaturonase (PG), β-glucosidase (BG) and the expressions of PsPG1 and PsBG1 genes at the base of the petals absciss zone were reduced, which delayed the senescence process of cut tree peony flowers, thus significantly extended its vase life. Nevertheless, the exogenous CEPA significantly accelerated the release rate of endogenous ethylene in the petals, increased the activities of IAA oxidase (IAAO), PG and BG, accelerated the senescence of the absciss zone cells in the petals of cut flowers, and reduced the petal anti-falling ability, thus promoted its wilting and falling. Meanwhile, the vase life and the optimal viewing period of AVB+CEPA composite pretreatment were not much different from control, so CEPA could partially offset the physiological effects of AVB. 【Conclusion】 For the ethylene sensitive cut tree peony Luoyanghong, endogenous auxin was involved in the process of ethylene promoting the abscission of petal absciss zone cells. The control of ethylene was the basic way to improve the quality of Luoyanghong cut flowers.

Changes of Antioxidant Activity and Its Possible Mechanism in Tan Sheep Meat in Different Postmortem Time

HOU ChengLi,HUANG CaiYan,ZHENG XiaoChun,LIU WeiHua,YANG Qi,ZHANG DeQuan

Scientia Agricultura Sinica. 2021, 54(23): 5110-5124. doi:10.3864/j.issn.0578-1752.2021.23.015

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【Objective】The aim of this study was to explore the changes of antioxidant activity of Tan sheep meat at different time points after slaughter, and to explain its mechanism from the perspective of free amino acids and proteome, so as to provide the data support for the quality maintenance of fresh mutton. 【Method】Six 6-month-old healthy stall-feeding male lambs (18.30±1.41 kg) were selected in this study. The longissimus dorsi of the lambs was collected at 0.5, 3, 6, 12 and 48 h after slaughter to determine the ferric ion reducing antioxidant power (FRAP), free radical scavenging capacity of 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH), oxygen-radical absorbance capacity (ORAC) of DPPH, total antioxidant capacity of N,N-dimethyl-p-phenylendiamine (DMPD), content of free amino acids and the changes of proteomic. 【Result】The results showed that within 48 h postmortem, the FRAP total antioxidant capacity decreased first and then increased. While the free radical scavenging capacity of ABTS increased first and then tended to be stable. The free radical scavenging capacity and ORAC of DPPH and DMPD all showed an upward trend. Total antioxidant capacity of FRAP, free radical scavenging capacity and ORAC of ABTS, DPPH, DMPD of Tan sheep meat at 48 h after slaughter were all significantly higher than those at 0.5 and 3 h after slaughter (P<0.05). No significant difference was found in total content of free amino acids (P>0.05), but the contents of Cys, Leu, Tyr, Ile, Phe and Arg in Tan sheep meat were significantly different among different time points after slaughter (P<0.05), and showed an increase trend. There were 7 free amino acids positively correlated with FRAR, including o-phosphoserine (r = 0.489, P<0.01), leucine (r=0.566, P<0.01), tyrosine (r=0.596, P<0.01), isoleucine (r=0.374, P<0.05), phenylalanine (r=0.499, P<0.01), lysine (r=0.375, P<0.05) and arginine (r=0.376, P<0.05). Among them, Leu, Tyr, Phe and Arg were significantly positively correlated with FRAP, ABTS, DPPH, ORAC and DMPD (P<0.05). Twenty differential proteins in Tan sheep meat at different time points after slaughter were screened and identified by proteomic. There were 12 differential proteins significantly correlated with FRAP (P<0.05). Calpain inhibitor protein and unknown protein 2 were significantly negatively correlated with FRAP, ABTS, DPPH, ORAC and DMPD (P<0.05). Glycogen protein 1 and protein kinase domain proteins were significantly positively correlated with FRAP, ABTS, DPPH, ORAC and DMPD (P<0.05). 【Conclusion】This research indicated that the total antioxidant capacity of Tan sheep meat increased within 48 h after slaughter, which was related to the release of free amino acids such as Leu, Tyr, Phe and Arg in the postmortem muscle. The decrease protein expression of calpain inhibitor protein and unknown protein 2, and the increased protein expression of glycogen protein 1 and protein kinase domain protein in muscle were significantly correlated with the changes of antioxidant capacity of Tan sheep meat after slaughter.

Comparison of Genomic Prediction Accuracy for Meat Type Chicken Carcass Traits Based on GBLUP and BayesB Method

ZHU Mo,ZHENG MaiQing,CUI HuanXian,ZHAO GuiPing,LIU Yang

Scientia Agricultura Sinica. 2021, 54(23): 5125-5131. doi:10.3864/j.issn.0578-1752.2021.23.016

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【Objective】 Predicting the breeding value is the core content of livestock breeding and the accurate predicting of breeding value is an important approach to improve the selection accuracy of breeding. Carcass traits are economic important traits for broilers. However, carcass traits can only be measured postmortem. Genomic selection may be a powerful tool because of its accurate prediction of breeding values of animals without own phenotypic information. At present, there are few reports on genomic selection on carcass traits for broilers. The purpose of this study was to compare the accuracy of genomic prediction of broiler carcass traits by using GBLUP and BayesB method. 【Method】 This study investigated the efficiency of genomic prediction in a white-feathered broiler population, which was collected from 3 362 white-feathered broilers. Five carcass traits, including breast muscle rate (BrR), breast muscle weight (BrW), carcass weight (CW), thigh muscle rate (ThR) and thigh muscle weight (ThW) were taken into account. All the individuals were genotyped with “IASCHICK” chicken 55 K SNP array. PLINK software was used for the quality control of genotype data. GBLUP and BayesB method were implemented with ASReml and hibayes package in R software, respectively. Generation validation were utilized to evaluate the accuracy of genomic prediction for twenty replicates for each trait.【Result】The results showed that the accuracy of genomic selection was almost positively correlated with the heritability of each trait. The validation results indicated that the prediction accuracy of BrR was the highest with GBLUP method, and the accuracies of BrR, BrW, CW, ThR and ThW were 0.3262, 0.2871, 0.2780, 0.2153, and 0.2126, respectively. Meanwhile, the accuracy of BrR was the highest with BayesB method, and the accuracies of BrR, BrW, CW, ThR and ThW were 0.3765, 0.2257, 0.1376, 0.2525, and 0.2844, respectively. The results showed that the prediction accuracy of BayesB method was slightly higher than that of GBLUP method. It took about 1 h and 7 h for GBLUP and BayesB method, respectively, to carry out the calculating procedure for one trait. 【Conclusion】 In conclusion, the prediction accuracy of BrR, ThR and ThW with BayesB method was higher than that with GBLUP method, while the prediction accuracy of BrW and CW with GBLUP method was higher than that with BayesB method. However, the calculating time for BayesB method was longer than that for GBLUP method. In breeding practice, the balance of prediction accuracy and computational efficiency should be comprehensively considered to predict the genomic estimated breeding value.

Effects of miR-31-5p on the Proliferation and Apoptosis of Hair Follicle Stem Cells in Goat

FENG YunKui,WANG Jian,MA JinLiang,ZHANG LiuMing,LI YongJun

Scientia Agricultura Sinica. 2021, 54(23): 5132-5143. doi:10.3864/j.issn.0578-1752.2021.23.017

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【Objective】 The Yangtze River Delta White Goat is the only goat breed that can produce superior-quality brush hair in China and the world. The transcriptome sequencing results of the research team showed that there were significant differences in the expression level of MAP3K1 in the individual skin tissues of superior-quality brush hair and normal-quality brush hair. This study aimed to explore the key miRNAs that interacted with MAP3K1 during the formation of superior-quality brush hair and their effects on the proliferation and apoptosis of goat hair follicle stem cells. 【Method】 The bioinformatics websites (StrBase, miRDB, TargetScan, miRWalk, DAVID, KEGG, and RNAhybrid) were used to predict and select the miRNAs, with targeted relationship with MAP3K1, and use the online website Venny 2.1 to draw a Venn diagram. Through the construction of miR-31-5p overexpression vector, wild-MAP3K1, wild/Mut-RASA1 Luciferase Reporter assay vector, the relationship between miR-31-5p and MAP3K1, RASA1 was verified, and the effects of miR31-5p on MAP3K1, RASA1 mRNA and protein expression were detected by qPCR and Western Blot. In order to explore the effect of overexpression of miR-31-5p on cell proliferation and apoptosis, the mRNA and protein expression levels of proliferation marker gene (PCNA,CDK1,CCND2), anti-apoptotic gene (Bcl-2) and pro-apoptotic gene (Bax) in hair follicle stem cells transfected with miR-31-5p were detected, and the effects of overexpression of miR-31-5p on the viability, cell cycle and apoptosis of hair follicle stem cells were verified by CCK-8, EdU, flow cytometry. 【Result】 Through the database, the final score of the three miRNAs were predicted, which might relatively highly interact with MAP3K1. Then, combined with the known miRNAs studies in skin and hair follicle cells, miR-31-5p with highest score was selected as research object. After transfection of miR-31-5p, the relative expression of miR-31-5p in cells was detected, and it was found that the expression of miR-31-5p was significantly higher than that in the control group and blank vector group (P<0.01). The results of double luciferase reporter genes showed that overexpression of miR-31-5p could increase the activity of MAP3K1. Combined with Target Scan and KEGG database, it was predicted that miR-31-5p could target RASA1, the upstream inhibitor of MAP3K1 in MAPK signal pathway. In order to verify the relationship between miR-31-5p and RASA1, it was found that overexpression of miR31-5p inhibited the activity of RASA1 (P<0.01); qPCR and Western Blot assays showed that overexpression of miR-31-5p significantly inhibited the expression of mRNA and protein of RASA1 and promoted the expression of MAP3K1 (P<0.01). CCK-8 assays showed that overexpression of miR-31-5p increased the ability of cell proliferation. EdU staining showed that the rate of positive cells overexpressing miR-31-5p was significantly higher than that in the blank group (P<0.01), and promoted cell proliferation. Cell cycle data showed that after overexpression of miR-31-5p, the proportion of cells in G1/G0 phase was 52.23%, which was significantly lower than that in Control group (56.81% P<0.01). It slowed down the cell arrest in G1/G0 phase, but there was no significant difference between S phase and G2/M phase, however there was still an upward trend. Through apoptosis experiment, it was found that the survival rate of miR-31-5p group was 93.8%, and the total apoptosis rate was 4.9%; while that of control group was only 90.1%, and the total apoptosis rate was 8.41%, which indicated that the apoptosis rate decreased significantly after overexpression of miR-31-5p. Finally, the effects of miR-31-5p on proliferation and apoptosis-related genes were detected. It was found that overexpression of miR-31-5p significantly increased the mRNA and protein expression levels of proliferation marker genes and anti-apoptosis genes (Bcl-2), and decreased the mRNA and protein expression levels of pro-apoptosis gene (Bax). According to the results of the research, the molecular mechanism of miR-31-5p in hair follicle stem cells was revealed. 【Conclusion】 miR-31-5p targeted RASA1 and up-regulated the expression level of MAP3K1, thereby promoting the proliferation of hair follicle stem cells and inhibiting their apoptosis. It provided a theoretical basis for further investigating the molecular mechanism that regulates the characteristics of superior-quality brush hair of the Yangtze River Delta White Goats.

Biofilm-Forming Phenotype, Antibacterial Resistance Genes, Integrase Genes and Virulence Genes Detection of Escherichia coli Isolated from Yaks and Tibetan Pigs in Northwest Sichuan Plateau

CHEN ChaoXi,LI YuHan,TAN Min,WANG Lu,HUANG ZhiHong

Scientia Agricultura Sinica. 2021, 54(23): 5144-5162. doi:10.3864/j.issn.0578-1752.2021.23.018

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【Objective】To improve the understanding of biological properties of drug resistance status, virulence characteristics and the predominant phylogroups of Escherichia coli (E.coli) isolated from yaks and Tibetan pigs in northwest Sichuan Plateau, biofilm-forming ability, antibacterial resistance genes, virulence genes, integrase genes and phylogenetic analyses were carried out in current study.【Method】Fecal samples and gastrointestinal contents from yaks and Tibetan pigs were collected for E.coli isolation and identification using MacConkey agar and 15e enterobacteriaceae bacterial biochemical coding identification tube. Modified semi-quantitative crystal violet staining method and microdilution broth method were used for biofilm-forming ability and antibacterial sensitivity testing to 24 antibacterial agents, respectively. Meanwhile, the detection of 28 antibacterial resistance genes, 2 integrase genes and 15 virulence genes and phylogenetic analyses were performed by conventional PCR or multiple PCR method. 【Result】The results showed that: (1) 329 strains of E.coli were isolated and identified from 471 feces and gastrointestinal samples collected from yaks and Tibetan pigs, and the isolation rate was 78.9%. (2) Most of the 329 strains of E.coli performed weak or absent biofilm-forming ability, and only 2 strains showed strong biofilm formation phenotype (one isolated from yak and the other isolated from Tibetan pig). (3) Most of the 329 strains of E.coli revealed drug resistance to 24 antibacterial agents and were multi-drug resistant, among which, the drug resistance rates to Sulfamethoxazole, Sulfadimidine, Streptomycin, Chloramphenicol, Ampicillin, Rifampicin, and Oxytetracycline were relatively high, and were sensitive to Aminoglycosides (Kanamycin, Amikacin, Spectinomycin), β-lactams(Ceftiofur, Ceftriaxone, Cefazolin), Quinolones (Nalidixic acid, Sarafloxacin, Enrofloxacin, Ciprofloxacin, Danofloxacin, Levofloxacin) and Colistin B. (4) Twenty-one antibacterial resistance genes (ARGs) were detected positive and the other seven ARGs (cat1, cat2, bla_CMY-2, bla_SHV, tetC, tetG, and tetX) were detected negative, among which aac(6')-Ib was the most prevalent gene, followed by sul1 and floR, with the detection rates over 30%. There existed correlation between drug resistance to quinolones antibiotics and qnrA in Tibetan pig-derived E.coli, and for yak-derived E.coli, the strains resistant to β-lactam antibiotics existed correlation between bla_TEM and bla_DHA. (5) The detection rates of integrase genes intⅠ1 and intⅠ2 were 30.09% (99/329) and 4.56% (15/329), respectively. And integrase genes intⅠ1 and intⅠ2 were simultaneously detected in 10 isolates (2 yak-derived and 8 Tibetan pig-derived, respectively). (6) Virulence genes of agn43, sitA, ompT, eaeA, bcsA, fimC, LT, fyuA and irp2 were all positively detected, but stx1, stx2, ehxA, bcsB, hlyA, and hlyE were not detected. There existed 38 different virulence genotypes in 329 strains of E.coli and 285 of which carried at least one of the seven virulence genes except for agn43 and bcsA, some strains carried six virulence genes at most. (7) Among the 21 ARGs, types of ARGs in phylogroup A and B1 were more abundant than those of phylogroup B2 and D; In phylogroup A, sul3, qnrS, tetM were the most widely distributed ARGs, and for phylogroup B1 most widely distributed ARGs were sul1 and aac(6')-Ib, without tetM and qnrA; Seven virulence genes were mainly distributed in phylogroup A and B1, fimC, sitA and ompT genes were mainly distributed in phylogroup A and B1, and eaeA, fyuA and irp2 were the mainly distributed genes in phylogroup B1; LT was mainly distributed in phylogroup A (only one distributed in phylogroup D).【Conclusion】 In summary, the resistance status of 329 strains of E.coli was serious, revealing various drug resistance profiles and virulence genotypes. The current study could provide data support and theoretical basis for yak and Tibetan pig colibacillosis treatment, mechanism of pathogenesis and rational use of antibacterial agents in northwest Sichuan Plateau.

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